Since our first report of darunavir (DRV) in 2003, we continued optimization based on the structure of DRV, seeking novel protease inhibitors (PIs) that are more potent against a variety of existing multi-PI-resistant HIV-1 variants with greater safety, do not permit or substantially delay the emergence of HIV-1 variants resistant to the very PIs, and favorably penetrate into the CNS, and identified GRL-142. GRL-142 contains newly generated pharmacophores such as an unprecedented 6-5-5 ring-fused crown-like tetrahydropyranofuran as the P2-ligand (Crn-THF), P1-bis-fluoro-benzyl (bis-Fbz), and P2'-cyclopropyl-amino-benzothiazole (Cp-Abt). GRL-139, a prototype to GRL-142, structurally resembles DRV but contains the Crn-THF moiety instead of the bis-THF of DRV, and exerts comparable antiviral activity against wild-type HIV-1 (HIVNL4-3) as compared to DRV. However, GRL-139 failed to block the replication of three highly DRV-resistant HIV-1 variants (HIVDRVRs) that were selected by propagating in the presence of increasing concentrations of DRV and are highly resistant to all presently clinically available PIs including DRV and nucleos(t)ide-reverse-transcriptase inhibitors such as tenofovir (TDF). By contrast, GRL-036 also resembles DRV but has the Cp-Abt moiety and shows an improved anti-HIV-1 profile, more effectively blocking the replication of HIVDRVRs than DRV. GRL-121 contains both Crn-THF and Cp-Abt moieties and more effectively blocked the replication of HIVNL4-3 by about 10-fold compared to DRV. GRL-121 more potently suppressed the replication of all three HIVDRVRs. Interestingly, the addition of two fluorine atoms to GRL-121, generating GRL-142 further strengthened the activity against HIVNL4-3 achieving an IC50 value as low as 0.019 nM compared to the 9 FDA-approved PIs of which IC50s range from 3.2 to 330 nM. GRL-142 had a much improved cytotoxicity profile with a selectivity index (CC50/IC50) as high as 2,473,684. GRL-142 also highly potently blocked the replication of all three HIVDRVRs by factors of 27-83 compared to GRL-121. Notably, the IC50 value of GRL-142 against HIVDRVRP51 (1.2 nM), the most multi-PI/NRTI-resistant HIVDRVR, was 3-fold lower than that of DRV against HIVNL4-3 (3.2 nM). We further examined the activity of GRL-142 against two HIV-2 strains and found that GRL-142 also exerts highly potent antiviral activity against the HIV-2 strains examined. We further examined the activity of five PIs including GRL-121 and -142 against seven resistant HIV-1 variants, which we had previously selected in vitro with each of the seven FDA-approved PIs (invitroHIVPIRs). Most of the seven variants were significantly less susceptible to two PIs, lopinavir (LPV) and ATV, that have presently been relatively well used in clinics. DRV also failed to effectively block most of the seven variants with IC50 value fold-differences ranging from 2- to 86-fold. However, GRL-121 showed extremely potent activity against all the seven variants examined, presenting IC50 values ranging 0.0018 to 0.13 nM. The activity of GRL-121 against all of the seven variants was significantly more potent than that against HIVNL4-3. Surprisingly, GRL-142 showed even further more potent activity against the seven variants with IC50 values of 0.0000019 nM (1.9 fM) to 0.015 nM. The activity of GRL-142 against the variants was also significantly more potent than that against HIVNL4-3. We have previously demonstrated that PR monomer subunits initially interact at the active site interface, generating unstably dimerized PR subunits, and subsequently the termini interface interactions occur, completing the dimerization process. DRV binds in the proximity of the active site interface of PR and blocks PR subunits dimerization. Therefore, we asked whether GRL-142 binds to monomer subunits using electrospray ionization mass spectrometry (ESI-MS). As shown in Figure 2C, the ESI-MS spectra of PR containing D25N substitution (PRD25N), which was folded in the presence of drugs revealed five peaks of differently charged ions in the range of mass/charge ratio (m/z) of 1,500-2,900. Since +5 charged monomer ion and +10 charged dimer ion have the same m/z (m/z=2164.75 for PRD25N), the greatest peak detected at m/z 2164.75 was determined to represent two forms, a PR monomer and PR dimer. Thus, the five peaks represent a monomer, two dimers, and two monomer+dimers. When unfolded PRD25N was re-folded in the presence of DRV, six additional significant peaks appeared, three for monomer+DRV, and three for dimer+DRV. When the same PRD25N was re-folded in the presence of GRL-142, six significant peaks appeared, representing three for monomer+GRL-142, and three for dimer+GRL-142. Each of the six additional peaks seen with GRL-142 appeared greater than those seen with DRV. When compared with the heights of the dimer+monomer peak rendered 1.0, the average height of the three peaks of DRV-bound monomers and that of the three peaks of GRL-142-bound monomers were 0.046 and 0.312, respectively; and the average height of the three peaks with DRV-bound dimers and that of the three peaks with GRL-142-bound dimers were 0.060 and 0.188, respectively. These data suggest that GRL-142 more tightly bound to monomers by 6.78-fold and to dimers by 3.13-fold than DRV and at least in part explain the reason GRL-142 much more strongly blocked PR dimerization than DRV. Persistent HIV-1 replication and inflammation in the CNS, which can occur even in patients receiving cART with an undetectable plasma viral load, is most likely responsible for HAND. Hence, we finally quantified GRL-142 concentrations in plasma, cerebrospinal fluid (CSF), and brain of rats (n=2) and compared those figures with those of DRV obtained under the same conditions. When DRV was perorally administered at a dose of 5 mg/kg together with RTV (8.33 mg/kg), the Cmax was achieved around 90 min after the PO administration. The DRV concentrations determined in 15 and 90 min after the peroral administration turned out to be 0.595 microM and 0.847 microM in plasma; 0.00100 microM and 0.00116 microM in CSF; and 0.0110 microM and 0.0157 microM in brain, respectively. The plasma concentrations of DRV were much greater than the IC50 value (3.2 microM); however, concentrations in CSF were lower than the IC50 value and those in brain were slightly above the DRV IC50 value but still substantially lower than the DRV IC95 value of DRV (0.3 microM), suggesting that DRV likely fail blocking the replication of HIV-1 in the CNS. By contrast, when GRL-142 was perorally administered at a dose of 5 mg/kg together with RTV (8.33 mg/kg), the Cmax was achieved around 360 min after the PO administration. Plasma samples collected 60 and 360 min after the PO administration contained 0.189 microM GRL-142 and 0.974 microM, respectively. CSF samples contain below detection levels and 0.000532 microM); brain contained 0.00724 microM and 0.0326 microM in 60 and 360 min, respectively. Since the IC50 and IC95 values of GRL-142 are 19 pM and 0.28 nM, GRL-142 concentrations in brain are calculated to be 1,882-fold greater than the IC50 value of GRL-142 and 114-fold greater than IC95 of GRL-142, while 562-fold lower than CC50 of GRL-142. These data strongly suggest that GRL-142 would potently block the infection and replication of HIV-1 in the brain.